The major source of energy for myocardial function is the oxidation of fatty acids in cardiac mitochondria. Our long range goal is to define mitochondrial biogenesis, particularly the coordinate regulation of nuclear genes encoding mitochondrial proteins, because expression of these genes changes during development and differs among tissues with various energy requirements. The cardiovascular system is an ideal model to explore regulation of nuclear genes encoding mitochondrial proteins because (i) of the heart's extraordinary energy requirements and (ii), during the transition from fetal to post-natal life, the mammalian heart switches from anaerobic, glycolytic energy production to aerobic oxidative phosphorylation with fatty acids as the major energy source. We postulate that a set of transcription factors regulates the coordinated changes in mitochondrial protein gene expression which occur with the rapid induction of mitochondrial number in the perinatal period. Our proposed focus is regulation of the two human mitochondrial creatine kinase (MtCK) genes. The ubiquitous MtCK gene is expressed in many tissues including vascular smooth muscle, but the sarcomeric MtCK gene is expressed only in heart and skeletal muscle. We will test the creatine phosphate (CP) shuttle hypothesis, which proposes that energy produced in mitochondria is transferred as CP to the myofibrillar apparatus and serves as the source of energy for myocardial contraction. Our specific aims are to (1) delineate tissue-specific regulatory sequences in the human MtCK genes by transient transfection in vitro; (2) characterize changes in endogenous mouse uMtCK and sMtCK gene expression during development by in situ hybridization and immunocytochemistry; (3) test putative tissue- specific regulatory elements for both the uMtCK and sMtCK genes in transgenic mice and by direct myocardial injection; (4) characterize regulatory elements mediating changes in expression of the uMtCK and sMtCK genes during development with transgenic mice; (5) determine the functional roles of the uMtCK and sMtCK genes by disruption of both mouse MtCK genes with homologous recombination to definitively test the CP shuttle hypothesis; and (6) Isolate and characterize transcription factors which bind to regulatory regions in the human sMtCK and uMtCK genes. Understanding regulation of expression of nuclear genes encoding these cardiac mitochondrial energy-producing proteins is essential to defining normal mammalian cardiovascular development and the pathophysiology of hypoxia (as in cyanotic congenital heart disease), hypertrophy, and ischemia and to delineating molecular defects in mitochondrial proteins which cause cardiomyopathy and sudden infant death.